Irina Poliakova Eide

A transcriptional profile of the decidua in preeclampsia

OBJECTIVE We sought to obtain insight into possible mechanisms underlying preeclampsia using genomewide transcriptional profiling in decidua basalis. STUDY DESIGN Genomewide transcriptional profiling was performed on decidua basalis tissue from preeclamptic (n = 37) and normal (n = 58) pregnancies. Differentially expressed genes were identified and merged into canonical pathways and networks. RESULTS Of the 26,504 expressed transcripts detected, 455 were differentially expressed (P < .05; false discovery rate, P < .1). Both novel (ARL5B, SLITRK4) and previously reported preeclampsia-associated (PLA2G7, HMOX1) genes were identified. Pathway analysis revealed that tryptophan metabolism, endoplasmic reticulum stress, linoleic acid metabolism, notch signaling, fatty acid metabolism, arachidonic acid metabolism, and NRF2-mediated oxidative stress response were overrepresented canonical pathways. CONCLUSION In the present study single genes, canonical pathways, and gene-gene networks that are likely to play an important role in the pathogenesis of preeclampsia have been identified. Future functional studies are needed to accomplish a greater understanding of the mechanisms involved.

Serious foetal growth restriction is associated with reduced proportions of natural killer cells in decidua basalis

Extravillous trophoblasts are major participants in placental development and remodelling of spiral arteries. Trophoblast invasion is regulated by maternal immune cells, and abnormal leucocyte subpopulation composition has been reported in implantation failure. In pre-eclampsia (PE), with or without foetal growth restriction (FGR), superficial trophoblast invasion and insufficient remodelling of spiral arteries are common findings. In the present study, we have compared spiral artery remodelling and leucocyte composition in decidual tissue from 30 cases (PE=8, FGR=5, PE + FGR=17) and 31 controls. Six histological remodelling criteria were established, and each pregnancy obtained a remodelling score. Numbers of natural killer (NK) cells (CD56+), T cells (CD3+) and activated (CD25+ or CD69+) leucocytes were determined and related to total leucocyte (CD45+) numbers in serial sections. Cases demonstrated significantly impaired spiral artery remodelling, inappropriate placental growth and reduced NK cell proportions, as compared to controls (P=0.02, P<0.001 and P=0.01, respectively). Reduced NK cell proportion was primarily found in pregnancies complicated by FGR, with or without PE, and a significant positive correlation was observed between NK cell proportion, trophoblast infiltration and placental growth. Our in vivo observations support the hypothesized association between NK cells, impaired placental development and pathogenesis of PE/FGR.

Whole-genome microarray and targeted analysis of angiogenesis- regulating gene expression (ENG, FLT1, VEGF, PlGF) in placentas from pre-eclamptic and small-for-gestational-age pregnancies

Objective. To compare the placental pathology associated with pre-eclampsia (PE) and/or fetal growth restriction, the transcriptomes of placental tissues from PE and small-for-gestational-age (SGA) pregnancies were explored. In addition, a targeted analysis of angiogenesis-regulating gene expression was performed. Methods. Whole-genome microarray analysis was performed on placental tissue from gestational age-matched PE (n = 10), SGA (n = 8) and PE + SGA (n = 10) pregnancies. The expression of genes regulating angiogenesis (endoglin (ENG), fms-related tyrosine kinase 1 (FLT1), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)) was analyzed by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). Results. Microarray analysis did not reveal any significant differences between groups. However, an increased expression of ENG and FLT1 was detected by qRT-PCR in the PE + SGA group. Conclusions. The placental transcriptome did not differ between groups, although an increased anti-angiogenic gene expression in PE + SGA was observed with qRT-PCR analysis. Based on this, we conclude that although microarray technology may represent a powerful tool in generating new hypothesis in complex fields, it may not be sensitive enough to detect subtle changes in gene expression.

Decidual expression and maternal serum levels of heme oxygenase 1 are increased in pre-eclampsia.

Background. Pre‐eclampsia (PE) is associated with increased oxidative stress and excessive maternal inflammatory response. Heme oxygenase 1 (HMOX1) is an important stress response enzyme and a mediator of cytoprotection against a wide variety of tissue injuries. Methods. In the present study, microarray technology was used to compare the expression of HMOX1 and other genes involved in stress and inflammatory responses in decidua basalis from 16 pregnancies complicated by PE and 17 healthy controls. In addition, the presence of HMOX1 protein in decidua basalis was examined by means of immunohistochemistry, and ELISA was used to measure the maternal serum concentration of HMOX1. Results. Fifteen transcripts involved in stress response including HMOX1 were up‐regulated in cases, using a cut‐off value at p = 0.01. HMOX1 protein expression in decidua basalis was significantly increased in cases compared to controls reflected by more pronounced intensity of HMOX1 positive decidual cells (1.8±0.3 versus 1.5±0.4, p = 0.02) and an increased proportion of HMOX1 positive decidual leukocytes (31±29 versus 9±6%, p = 0.001). Finally, serum HMOX1 levels were significantly higher among cases compared to controls (3.1±1.3 versus 1.9±0.5 ng/ml, p = 0.008). Conclusions. Increased decidual and serum HMOX1 levels, together with altered decidual expression of some stress‐related genes in cases, support the role of oxidative stress and excessive maternal inflammatory response in the pathogenesis of PE.

Matrix Metalloproteinase 1 in Pre-eclampsia and Fetal Growth Restriction: Reduced Gene Expression in Decidual Tissue and Protein Expression in Extravillous Trophoblasts

Superficial invasion of extravillous trophoblasts (EVTs) and impaired spiral artery remodelling are characterizing phenomena in pregnancies complicated by pre-eclampsia (PE) and fetal growth restriction (FGR). However, the underlying causes remain unclear. In this study, gene expression in decidua basalis tissue from pregnancies complicated with PE and/or FGR (n = 18) and normal pregnancies (n = 17) was assessed by Affymetrix HG Focus microarray to obtain hints of mechanisms involved in the pathogenesis. A total of 200 differentially expressed transcripts were detected at a false discovery rate (FDR) <or= 0.1. Several genes involved in trophoblast differentiation and invasion were downregulated, including the matrix metalloproteinases (MMPs) MMP1, -7 and -12. MMPs are a family of enzymes involved in degradation of extracellular matrix and have been ascribed a permissive role in trophoblast invasion. MMP1 had the highest fold change among the differentially expressed genes (four-fold downregulated) and was chosen for further investigation. Reduced MMP1 mRNA in decidual tissue was confirmed by RT-qPCR. MMP1 protein expression in EVTs was assessed by double immunofluorescence analysis, using antibodies against pro-MMP1 and cytokeratin 7. The proportion of MMP1 positive EVTs was reduced in all subgroups of cases (PE: n = 18, FGR: n = 11 and PE + FGR: n = 30) compared to controls (n = 23) (all ps < 0.05). Based on these findings, we hypothesize that reduced levels of MMP1 protein in EVTs could be linked to the impaired trophoblast invasion in PE and/or FGR.

Identification of ACOX2 as a shared genetic risk factor for preeclampsia and cardiovascular disease

Preeclampsia (PE) is a serious complication of pregnancy, which is highly correlated with later life cardiovascular disease (CVD). Many risk factors are common for both diseases, but the contribution of shared genes remains to be determined. In this study, we used an integrative strategy to assess lipid traits as risk factors for PE and CVD by whole genome transcriptional profiling performed on Norwegian decidua basalis tissues (N=95) from preeclamptic and normal pregnancies and on blood lymphocytes (N=1240) from the San Antonio Family Heart Study (SAFHS). Among 222 genes that were differentially expressed (false discovery rate (FDR) P-value <0.05) between the PE, cases and controls, we found one gene, ACOX2 (acyl-coenzyme A oxidase 2, branched chain), that was downregulated in PE whose transcription was also inversely correlated with triglyceride levels (P=5.6 × 10−7; FDR P-value=0.0002) in SAFHS. We further report associations between SNPs in the ACOX2 gene and the transcription level (P-value=0.0045) of the gene, as well as with triglyceride levels (P-value=0.0051). ACOX2 is involved in bile acid production, a process that has been associated with both oxidative stress and regulation of triglyceride levels. Oxidative stress and increased triglyceride levels are known risk factors for CVD and both have also been associated with PE. Our results suggest that downregulation of ACOX2 is a shared risk factor for PE and CVD.

STOX2 but not STOX1 is differentially expressed in decidua from pre-eclamptic women: data from the Second Nord-Trøndelag Health Study

Variation in the Storkhead box-1 (STOX1) gene has previously been associated with pre-eclampsia. In this study, we assess candidate single nucleotide polymorphisms (SNPs) in STOX1 in an independent population cohort of pre-eclamptic (n = 1.139) and non-pre-eclamptic (n = 2.269) women (the HUNT2 study). We also compare gene expression levels of STOX1 and its paralogue, Storkhead box-2 (STOX2) in decidual tissue from pregnancies complicated by pre-eclampsia and/or fetal growth restriction (FGR) (n = 40) to expression levels in decidual tissue from uncomplicated pregnancies (n = 59). We cannot confirm association of the candidate SNPs to pre-eclampsia (P > 0.05). For STOX1, no differential gene expression was observed in any of the case groups, whereas STOX2 showed significantly lower expression in deciduas from pregnancies complicated by both pre-eclampsia and FGR as compared with controls (P = 0.01). We further report a strong correlation between transcriptional alterations reported previously in choriocarcinoma cells over expressing STOX1A and alterations observed in decidual tissue of pre-eclamptic women with FGR.

STOX2 but not STOX1 is differentially expressed in decidua from preeclamptic women

Variation in the Storkhead box-1 (STOX1) gene has previously been associated with pre-eclampsia. In this study, we assess candidate single nucleotide polymorphisms (SNPs) in STOX1 in an independent population cohort of pre-eclamptic (n = 1.139) and non-pre-eclamptic (n = 2.269) women (the HUNT2 study). We also compare gene expression levels of STOX1 and its paralogue, Storkhead box-2 (STOX2) in decidual tissue from pregnancies complicated by pre-eclampsia and/or fetal growth restriction (FGR) (n = 40) to expression levels in decidual tissue from uncomplicated pregnancies (n = 59). We cannot confirm association of the candidate SNPs to pre-eclampsia (P > 0.05). For STOX1, no differential gene expression was observed in any of the case groups, whereas STOX2 showed significantly lower expression in deciduas from pregnancies complicated by both pre-eclampsia and FGR as compared with controls (P = 0.01). We further report a strong correlation between transcriptional alterations reported previously in choriocarcinoma cells over expressing STOX1A and alterations observed in decidual tissue of pre-eclamptic women with FGR.

M9.4 Endoplasmatic reticulum stress in decidual tissue from pregnancies complicated by preeclampsia

Matthew Johnson1, John Blangero1, Christine East2, Rigmor Austgulen3, Shaun Brennecke2, Eric Moses1. 1Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX, USA; 2Department of Perinatal Medicine, Royal Women’s Hospital & Department of Obstetrics & Gynaecology, University of Melbourne, Melbourne, Australia; 3Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway